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WO2019047914A1 - Molécule d'arn double brin ciblant ckip-1 et son utilisation - Google Patents

Molécule d'arn double brin ciblant ckip-1 et son utilisation Download PDF

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WO2019047914A1
WO2019047914A1 PCT/CN2018/104552 CN2018104552W WO2019047914A1 WO 2019047914 A1 WO2019047914 A1 WO 2019047914A1 CN 2018104552 W CN2018104552 W CN 2018104552W WO 2019047914 A1 WO2019047914 A1 WO 2019047914A1
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nucleotide
group
dsrna molecule
dsrna
subject
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Inventor
赵焰平
王红军
姜媛媛
仲伟婷
庞建梅
李功
李想
何伊欣
周丽莹
刘亚男
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Beijing Tide Pharmaceutical Co Ltd
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Beijing Tide Pharmaceutical Co Ltd
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Priority to CN202311783979.XA priority Critical patent/CN117866959A/zh
Priority to CN201880057905.9A priority patent/CN111433360B/zh
Priority to US16/644,977 priority patent/US11155819B2/en
Priority to EP18853084.4A priority patent/EP3680334A4/fr
Publication of WO2019047914A1 publication Critical patent/WO2019047914A1/fr
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Priority to US17/474,396 priority patent/US11939578B2/en
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Definitions

  • the present invention relates to the field of biomedicine, in particular, the present invention relates to double-stranded RNA molecules targeting CKIP-1 and uses thereof, in particular to the use of said double-stranded RNA molecules for the treatment of inflammatory diseases, such as arthritis, especially rheumatoid joints. The use of inflammation.
  • CKIP-1 (casein kinase interacting protein 1) is a bone formation inhibitory gene that specifically regulates bone formation rather than bone resorption. CKIP-1 is highly expressed in bone tissue in patients with osteoporosis. Targeted inhibition of CKIP-1 expression has been shown to be useful in the treatment of osteoporosis or other pathological bone destruction. However, CKIP-1 has not been associated with inflammation in the prior art.
  • TNF- ⁇ and IL-6 are two important pro-inflammatory cytokines that play an important role in the body's inflammatory response. Under physiological conditions, levels of TNF- ⁇ and IL-6 are low in the human body. However, under pathological conditions, an increase in the secretion of TNF-[alpha] and IL-6 and the resulting cascade of various pro-inflammatory factors may result in an inflammatory response leading to tissue damage. Inflammatory diseases have been treated in the art by targeted inhibition of TNF-[alpha] and IL-6. For example, inhibitors targeting TNF-[alpha] have been marketed in several varieties, including infliximab, etanercept, adalimumab, golimumab, and certolizumab. In addition, IL-6 blockers have been approved for clinical use, such as tocilizumab. In large randomized, double-blind clinical trials, tocilizumab has a better therapeutic effect in patients who do not respond to TNF- ⁇ monoclonal antibody.
  • Rheumatoid arthritis is a chronic systemic autoimmune disease characterized by multiple joint synovial inflammatory lesions. Prolonged recurrent episodes of synovitis can lead to destruction of cartilage and bone in the joints, joint dysfunction, and even disability.
  • Rheumatoid arthritis has a high incidence in adults, with an incidence of about 20-40% per 100,000 adult population. Studies have shown that 70-75% of patients with rheumatoid arthritis can have bone destruction within 3 years of onset, 10% have severe dysfunction within 2 years of onset, and about 50% of patients lose their ability to work after 10 years of illness. It poses a serious financial burden on patients and society.
  • the drugs for treating RA mainly include non-steroidal anti-inflammatory drugs, hormones, anti-rheumatic drugs, etc., which are mainly used to relieve pain, reduce inflammation, and can not well prevent joint and bone destruction.
  • Some new biological agents have emerged in recent years to alleviate and inhibit the occurrence of bone destruction, but they cannot repair existing bone damage.
  • RA treatment drugs that can reduce inflammation and promote bone repair.
  • the invention provides a double stranded RNA (dsRNA) molecule comprising a sense strand and an antisense strand selected from the group consisting of:
  • the sense strand and/or antisense strand additionally has an overhang of at least one nucleotide at the 3' end. In some embodiments, the sense strand and/or antisense strand additionally has a 2 nucleotide overhang at the 3' end, preferably the overhang is TT.
  • the substitutions are located at 5, 5, 4, 3 or 2 of the 5' and/or 3' end Within the nucleotides. In some embodiments, wherein the sense strand and the anti-sense strand comprise 1 nucleotide substitution, the substitution being located at the 3' last nucleotide position of the sense strand and correspondingly the 5' end of the antisense strand A nucleotide position.
  • the dsRNA comprises at least one modified nucleotide.
  • the modified nucleotide is selected from the group consisting of: a 2'-O-methyl modified nucleotide, a 2'-F modified nucleotide, and a 5'-phosphorothioate group.
  • Nucleotide and terminal nucleotide 2'-deoxy-2'-fluoro modified nucleotide, 2'-deoxy-modified nucleoside linked to a cholesteryl derivative or a dodecanoic acid biguanide group Acid, locked nucleotide, abasic nucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide, morpholino nucleotide, phosphoramidate and non-tron base Base nucleotide.
  • the 2' hydroxyl group of the nucleotide of all uracil bases or cytosine bases in the sense strand and/or the antisense strand of the dsRNA is modified by a methoxy group.
  • the dsRNA molecule is siRNA or shRNA. In some embodiments, the dsRNA molecule inhibits expression of CKIP-1 by at least 50%, preferably by at least 70%. In some embodiments, the dsRNA molecule inhibits expression of pro-inflammatory cytokines such as IL-6, TNF-[alpha] and/or IL-17A.
  • the invention also provides an expression vector comprising a nucleotide sequence encoding a dsRNA molecule of the invention operably linked to a transcriptional regulatory element.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a dsRNA molecule of the invention or an expression vector of the invention, and a pharmaceutically acceptable carrier.
  • the present invention provides a method of treating arthritis, particularly rheumatoid arthritis, in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a dsRNA molecule of the invention or an expression vector of the invention Or a pharmaceutical composition of the invention.
  • the method further comprises administering to the subject an additional therapeutic agent for treating arthritis, particularly rheumatoid arthritis.
  • the present invention provides a dsRNA molecule of the present invention or an expression vector of the present invention or a pharmaceutical composition of the present invention, in the preparation of a medicament for treating arthritis, particularly rheumatoid arthritis, in a subject in need thereof use.
  • the invention provides a method of treating an inflammatory disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a dsRNA molecule of the invention or an expression vector of the invention or a pharmaceutical composition of the invention .
  • the method further comprises administering to the subject an additional therapeutic agent for treating an inflammatory disease.
  • the invention provides the use of a dsRNA molecule of the invention or an expression vector of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament for the treatment of an inflammatory disease in a subject in need thereof.
  • the present invention provides a method of treating a bone metabolism-related disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a dsRNA molecule of the present invention or an expression vector of the present invention or a medicament of the present invention combination.
  • the method further comprises administering to the subject an additional therapeutic agent for treating a disease associated with bone metabolism.
  • the invention provides the use of a dsRNA molecule of the invention or an expression vector of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament for the treatment of a disease associated with bone metabolism in a subject in need thereof.
  • Figure 1 shows a pool of candidate siRNA sequences.
  • the TT at the 3' end of each sequence is a dangling that is not complementary to the target sequence.
  • Figure 2 shows a vector map of an overexpression vector for dual luciferase assay.
  • Figure 3 shows the inhibitory effect of si-TD137 on CKIP-1 expression in a dual luciferase assay.
  • Figure 4 is a graph showing the inhibitory effect of si-TD141 on CKIP-1 expression in a dual luciferase assay.
  • Figure 5 shows the inhibitory effect of si-TD176 on CKIP-1 expression in the dual luciferase assay.
  • Figure 6 shows the inhibitory effect of si-7 on CKIP-1 expression in the dual luciferase assay.
  • Figure 7 Shows that siRNA reduces CIA mouse clinical score.
  • Figure 8 shows changes in body weight of CIA mice after siRNA treatment.
  • Figure 9 Shows that siRNA affects pro-inflammatory cytokine expression in joint tissues of CIA mice.
  • nucleic acid refers to DNA or RNA or a modified form thereof comprising a purine or pyrimidine base present in DNA (adenine "A”, cytosine “C”, guanine “G”, thymine “T ”) or a purine or pyrimidine base present in RNA (adenine "A”, cytosine "C”, guanine “G”, uracil “U”).
  • the double-stranded RNA nucleic acid molecules provided herein can also comprise a "T” base at the 3' end, even though the "T" base is not naturally occurring in the RNA. In some cases, these bases can be represented as "dT” to distinguish between deoxyribonucleotides present in the ribonucleotide chain.
  • a gene is "targeted" by a nucleic acid molecule of the invention when the nucleic acid molecule selectively reduces or inhibits expression of the gene.
  • a nucleic acid molecule targets the gene when it hybridizes under stringent conditions to a gene transcript (ie, its mRNA).
  • Hybridization "under stringent conditions” means annealing to a target mRNA region under standard conditions that tend to be detrimental to hybridization, for example, high temperature and/or low salt content.
  • a suitable procedure including 0.1 x SSC, 68 ° C, 2 hours) is described in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1982.
  • CKIP-1 refers to the CKIP-1 gene or protein (also known as PLEKHO1).
  • sequences of CKIP-1 include, but are not limited to, human: Genbank No. NM_016274.4; mouse: Genbank No. NM_023320.2; rat: Genbank No. NM_001025119.1 and cynomolgus monkey: Genbank No. XM_001098879 and XM_001098774.
  • target sequence refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the CKIP-1 gene, including mRNA of the RNA processing product of the primary transcript.
  • first nucleotide sequence when used to describe the relationship between a first nucleotide sequence and a second nucleotide sequence, unless otherwise indicated, refers to an oligonucleotide comprising the first nucleotide sequence or
  • first nucleotide sequence refers to an oligonucleotide comprising the first nucleotide sequence or
  • such conditions can be stringent conditions, wherein the stringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, at 50 ° C or 70 ° C for 12-16 hours, followed by washing.
  • Other conditions may also be applied, such as physiologically relevant conditions that may be encountered in a living organism. Those skilled in the art will be able to determine the range of conditions most suitable for the two-sequence complementation assay based on the final application of the hybrid nucleotide.
  • oligonucleotide when two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs should not be considered a mismatch when referring to a complementary definition.
  • the longer oligonucleotide comprises a shorter oligonucleotide A fully complementary 19 nucleotide sequence, which may also be referred to as "fully complementary.”
  • the "complementary" sequences used herein may also comprise or consist entirely of base pairs formed by non-Watson-Crick base pairs and/or formed from non-natural and modified nucleotides.
  • These non-Watson-Crick base pairs include, but are not limited to, G: U Wobble or Hoogstein base pairing.
  • a polynucleotide that is "substantially complementary" to at least a portion of a messenger RNA refers to a target mRNA comprising a 5' UTR, an open reading frame (ORF) or a 3' UTR (eg, an mRNA encoding CKIP-1)
  • mRNA messenger RNA
  • ORF open reading frame
  • 3' UTR eg, an mRNA encoding CKIP-1
  • a contiguous portion of a substantially complementary polynucleotide is complementary to at least a portion of CKIP-1 if the sequence of the polynucleotide is substantially complementary to a non-interrupting portion of the mRNA encoding CKIP-1.
  • Double-stranded RNA molecules have recently been found to block gene expression through a highly conserved regulatory mechanism known as RNA interference (RNAi).
  • RNAi RNA interference
  • WO 99/32619 discloses the use of dsRNAs of at least 25 nucleotides in length to inhibit expression of C. elegans genes. It has also been discovered that dsRNAs degrade target RNAs in other organisms, including plants (see, for example, WO 99/53050, Waterhouse et al. and WO 99/61631, Heifetz et al.), fruit flies (see, for example, Yang, D. et al., Curr. Biol.
  • double-stranded RNA refers to a duplex structure comprising two substantially parallel nucleic acid strands that are anti-parallel and as described above.
  • nucleotides of each chain are ribonucleotides, but as detailed herein, each strand or both strands may also comprise at least one non-ribonucleotide, such as deoxyribonucleotides and/or Modified nucleotide.
  • dsRNA as used in this specification may include chemical modifications to ribonucleotides, including modifications at multiple nucleotides, and includes all types of modifications disclosed herein or known in the art.
  • the two strands forming the duplex structure can be different portions of the same larger RNA molecule, or they can be separate RNA molecules. If the two strands are separate RNA molecules, such dsRNAs are often referred to in the literature as siRNAs ("short interfering RNAs"). If the two strands are part of one larger molecule and are joined by a non-interrupted nucleotide strand between the 3'-end of one strand forming the duplex structure and the 5'-end of the other strand, then The linked RNA strands are referred to as "hairpin loops", “short hairpin RNAs" or "shRNAs".
  • RNA strands can have the same or different number of nucleotides.
  • the dsRNA can comprise one or more nucleotide overhangs.
  • each strand or both strands may also comprise at least one non-ribonucleotide, eg, deoxyribonucleotides and/or Modified nucleotide.
  • &quot refers to one or more unpaired nucleotides that protrude from the duplex structure of a dsRNA when the 3' end of one strand of the dsRNA exceeds the 5' end of the other strand or vice versa.
  • "Flat end” or “blunt end” refers to the absence of unpaired nucleotides at the end of the dsRNA, ie, no nucleotide overhangs.
  • a “blunt-ended" dsRNA refers to a dsRNA that is double-stranded throughout its full length, i.e., has no nucleotide overhangs at either end of the molecule.
  • the chemical cap or non-nucleotide chemical moiety coupled to the 3'-terminus or 5'-end of the dsRNA is not considered when determining whether the dsRNA has a dangling or a blunt end.
  • antisense strand refers to a strand of a region of a dsRNA that comprises a region that is substantially complementary to a target sequence.
  • complementary region refers to a region of the antisense strand that is substantially complementary to a sequence (eg, a target sequence) as defined herein. If the complementary region is not completely complementary to the target sequence, the mismatch can be located in the interior or terminal region of the molecule. Generally, the most permissible mismatch is located in the terminal region (excluding the overhangs described herein), such as within the 5', 5, 4, 3 or 2 nucleotides of the 5' and/or 3' end, or is the most One nucleotide at the end.
  • sense strand refers to a strand of a dsRNA comprising a region that is substantially complementary to the antisense strand region.
  • subject or “individual” as used herein means a mammal, especially a primate, especially a human.
  • treating indicates partial or total relief of the symptoms of the individual, or remains unchanged after treatment.
  • treatment includes prevention, treatment, and/or cure.
  • Prevention refers to prevention of underlying disease and/or prevention of worsening symptoms or disease progression.
  • Treatment also includes any of the dsRNAs provided, expression vectors, and any pharmaceutical use of the compositions provided herein.
  • therapeutic effect refers to an effect caused by the treatment of an individual that alters, generally ameliorates or ameliorates the symptoms of the disease or condition, or cures the disease or condition.
  • therapeutically effective amount refers to an amount of a substance, compound, material, or composition comprising a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Thus, it is an amount necessary to prevent, cure, ameliorate, block or partially arrest the symptoms of a disease or condition. For example, if a given clinical treatment that reduces a measurable parameter associated with a disease or condition is considered to be an effective treatment, then a therapeutically effective amount of a medicament for treating the disease or condition is to reduce the parameter by at least 25%. The amount necessary.
  • pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent, such as a dsRNA.
  • a therapeutic agent such as a dsRNA.
  • Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • expression vector includes a vector capable of expressing a nucleotide sequence of interest operably linked to a regulatory sequence, such as a promoter region, capable of affecting expression of such a DNA fragment.
  • additional fragments can include promoter and terminator sequences, and can optionally include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like.
  • operably linked with respect to a nucleic acid sequence, region, element or domain means that the nucleic acid regions are functionally related to each other.
  • a promoter can be operably linked to a nucleotide sequence encoding a dsRNA such that the promoter regulates or mediates transcription of the nucleotide sequence.
  • nucleic acid molecule targeting CKIP-1 Second, the nucleic acid molecule targeting CKIP-1
  • the present inventors designed, synthesized and screened dsRNA molecules capable of significantly inhibiting the expression of CKIP-1.
  • the selected dsRNA molecules are both effective in reducing inflammation and promoting bone repair, and thus can be effectively used for the treatment of arthritis, such as rheumatoid arthritis (RA).
  • RA rheumatoid arthritis
  • the invention provides a nucleic acid molecule, such as a dsRNA molecule, that targets CKIP-1 comprising a sense strand selected from Table 1 and a corresponding complementary antisense strand.
  • the nucleic acid molecule that targets CKIP-1 comprises the corresponding ones in Table 1 corresponding to si-TD060, si-TD062, si-TD066, si-TD068, si-TD070, si-TD074, si-TD080 , si-TD082, si-TD089, si-TD096, si-TD137, si-TD140, si-TD141, si-TD143, si-TD176, si-TD178, si-TD181, si-TD362, si-TD364, si - Sense chain and antisense strand of TD378, si-TD726, si-TD730, si-7, si-10.
  • the nucleic acid molecule that targets CKIP-1 comprises the sense and antisense strands in Table 1 corresponding to si-TD137, si-TD141, si-TD176, si-7.
  • the sense strand and/or the antisense strand of the nucleic acid molecule additionally has a dangling of at least one nucleotide at the 3'
  • the sense strand and/or the antisense strand additionally have a dangling of 1, 2 or 3 nucleotides at the 3' end.
  • the overhang is TT (ie, dTdT).
  • the sense strand and the antisense strand of the nucleic acid molecule comprise an additional overhang TT at the 3' end.
  • the sense strand and/or the antisense strand in the nucleic acid molecule comprises at least one, for example, one or two nucleotide substitutions.
  • the substitution is within 6, 5, 4, 3 or 2 nucleotides of the 5' and/or 3' terminus.
  • the sense and antisense strands of the nucleic acid molecule comprise a nucleotide substitution at the 3' last nucleotide position of the sense strand and correspondingly the antisense strand 5' A nucleotide position. The substitution results in a mismatch to the target sequence, however the mismatch thus defined is permissible and does not significantly affect or affect the activity of the dsRNA.
  • a dsRNA of the invention comprises at least one modified nucleotide.
  • the modified nucleotide may comprise a modification of a phosphate group, a ribose group, and/or a base.
  • modification of a phosphate group in a nucleotide includes modification of the oxygen in the phosphate group, such as a Phosphorthioate modification and a Boranophosphate modification.
  • the oxygen in the phosphate group is replaced with sulfur and borane, respectively, as shown in the following formula. Both modifications stabilize the structure of the nucleic acid, maintaining high specificity and high affinity for base pairing.
  • Modification of the ribose group in the nucleotide includes modification of the 2'-hydroxy (2'-OH) in the ribose group.
  • 2'-OH When RNA is hydrolyzed, 2'-OH first attacks the phosphate group under catalysis by RNase, forms a cyclic phosphodiester while breaking the phosphoester bond, and forms a hydrolyzate under the action of a base.
  • the introduction of certain substituents such as methoxy or fluorine at the 2'-hydroxyl position of the ribose group provides nucleic acids such as siRNA with greater resistance to nuclease hydrolysis and increases the stability of the nucleic acid.
  • Modifications to the 2'-hydroxyl group of the pentose sugar include, but are not limited to, 2'-fluro modification, 2'-methoxy modification (2'-OME), 2'-methoxyethyl Modification (2'-MOE), 2'-2,4-dinitrophenol modification, LNA modification, 2'-Amino modification, 2 '-2'-Deoxy modification, 3'-cholesterol modification, 4'-thiothymidine modification, etc.
  • An example of the structure of such a modification is as follows:
  • the modification of a base in a nucleotide means that the base in the nucleotide group is modified, and the interaction of the base is enhanced by the modification of the base, thereby enhancing the effect on the target mRNA.
  • 5'-bromo-uracil and 5'-iodo-uracil modifications such as the introduction of bromine or iodine at the 5' position of uracil, are commonly used bases.
  • Modification method. Others include N3-methyl-uracil modification and 2,6-diaminopurine modification.
  • the dsRNA of the invention comprises at least one modified nucleotide selected from the group consisting of a 2'-O-methyl modified nucleotide, a 2'-F modified nucleotide, comprising 5' a nucleotide of a phosphorothioate group and a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid biguanide group, and/or, for example, the modified nucleotide is selected from the group consisting of Group: 2'-deoxy-2'-fluoro modified nucleotide, 2'-deoxy-modified nucleotide, locked nucleotide, abasic nucleotide, 2'-amino-modified nucleotide, 2 '-Alkyl-modified nucleotides, morpholino nucleotides, phosphoramidates, and nucleotides containing non-natural bases.
  • the modified nucleotide selected from
  • the at least one modified nucleotide can, for example, enhance the stability of the dsRNA and/or reduce the immunogenic effect of the dsRNA.
  • the modified nucleotide can be on the sense strand and/or on the antisense strand.
  • the dsRNA comprises at least one 2'-O-methyl modified ribonucleotide and/or at least one nucleotide comprising a 5'-thiophosphate group.
  • the 2' hydroxyl groups of the nucleotides of all uracil bases or cytosine bases in the sense strand and/or the antisense strand of the dsRNA of the invention are all modified by a methoxy group.
  • the 2' hydroxyl groups of all uracil bases or nucleotides of cytosine bases in the sense strand of the dsRNA of the invention are modified by a methoxy group.
  • the 2' hydroxyl groups of all nucleotides in the sense strand and/or antisense strand of the dsRNA of the invention are modified by a methoxy group.
  • the 2' hydroxyl groups of all nucleotides in the sense strand of the dsRNA of the invention are modified by a methoxy group.
  • the 5' end of the sense strand and/or the antisense strand of the dsRNA of the invention is phosphorylated.
  • the sense strand and/or antisense strand of a dsRNA of the invention comprises a 3' cholesterol modification.
  • the 2' hydroxyl group of the nucleotide group of all uracil bases or cytosine bases in the sense strand of the dsRNA of the invention is modified by fluorine (F).
  • the dsRNA of the invention comprises a locked nucleic acid modification in the sense strand.
  • all nucleotides in the sense strand and/or antisense strand of the dsRNA of the invention comprise a phosphorothioate modification.
  • the dsRNA molecule is an siRNA.
  • the dsRNA molecule is a shRNA (short hairpin RNA). It is within the ability of those skilled in the art to design suitable shRNA based on siRNA sequences.
  • the dsRNA of the present invention can be obtained by conventional techniques in the art such as solid phase synthesis or liquid phase synthesis. Modified nucleotides can be introduced by using modified nucleomonomers in the synthesis.
  • the invention provides an expression vector comprising a nucleotide sequence encoding a nucleic acid molecule of the invention, such as a dsRNA, wherein the nucleotide sequence is operably linked to a transcriptional regulatory element such as a promoter or the like.
  • a recombinant vector capable of expressing a dsRNA molecule can be delivered to a target cell and permanently present in the target cell.
  • a vector that provides transient expression of a nucleic acid molecule can be used.
  • the carrier can be administered repeatedly as needed. Once expressed, the dsRNA molecule interacts with the target mRNA and produces an RNA interference response.
  • shRNA is particularly suitable for production in this way.
  • the expression vector can be a linear construct, a circular plasmid vector or a viral vector (including but not limited to adenovirus, adeno-associated virus, lentiviral vector, etc.).
  • a single strand of siRNA can be transcribed from a promoter on two separate expression vectors; alternatively, each individual strand of siRNA can be transcribed from a promoter that is both located on the same expression plasmid.
  • shRNA the shRNA strand is transcribed from a single expression vector.
  • the promoter driving dsRNA expression in the expression vector of the present invention may be eukaryotic RNA polymerase I (for example, ribosomal RNA promoter), RNA polymerase II (for example, CMV early promoter or actin promoter or U1snRNA) Promoter) or generally an RNA polymerase III promoter (eg, U6snRNA or 7SKRNA promoter) or a prokaryotic promoter (eg, a T7 promoter, provided that the expression vector also encodes a T7 RNA polymerase required for transcription of the T7 promoter) ).
  • RNA polymerase I for example, ribosomal RNA promoter
  • RNA polymerase II for example, CMV early promoter or actin promoter or U1snRNA Promoter
  • RNA polymerase III promoter eg, U6snRNA or 7SKRNA promoter
  • a prokaryotic promoter eg, a T7 promoter, provided that the expression vector also encodes
  • the dsRNA of the present invention is capable of significantly inhibiting the expression of CKIP-1 in cells.
  • the expression of CKIP-1 is inhibited by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or even 100%.
  • the dsRNA of the invention is capable of inhibiting the expression of CKIP-1 by at least 50%. More preferably, preferably, the dsRNA of the invention is capable of inhibiting the expression of CKIP-1 by at least 70%.
  • inhibiting expression When the terms “inhibiting expression”, “down-regulating expression”, “repressing expression”, etc., are directed to the CKIP-1 gene, they are meant herein to at least partially inhibit the expression of the CKIP-1 gene by interacting with a second cell or cell.
  • the population is expressed as a decrease in the expression level of CKIP-1 in the first cell or cell population, wherein the CKIP-1 gene in the first cell or cell population is transcribed and it has been processed such that the expression of the CKIP-1 gene Inhibited, the second cell or population of cells is substantially identical to the first cell or population of cells but is not treated as such (control cells).
  • the degree of inhibition is usually expressed in the following way:
  • Control cell CKIP-1 expression level - treated cell CKIP-1 expression level (Control cell CKIP-1 expression level - treated cell CKIP-1 expression level) / Control cell CKIP-1 expression level x 100%
  • the expression level can be an mRNA level or a protein level. It is clear to those skilled in the art how to determine the mRNA level or corresponding protein level of a particular gene.
  • the dsRNA of the invention also inhibits the expression of the pro-inflammatory cytokines IL-6, TNF-[alpha] and/or IL-17A.
  • the dsRNA of the present invention can significantly inhibit the expression of the pro-inflammatory cytokine IL-6.
  • the expression of the pro-inflammatory cytokine IL-6, TNF- ⁇ , and/or IL-17A is inhibited by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60% , at least 70%, at least 80%, at least 90%, or even 100%. In some preferred embodiments, the expression of IL-6 is inhibited by at least 50%, more preferably by at least 80%.
  • the invention provides a pharmaceutical composition comprising at least one dsRNA of the invention or a nucleotide sequence expression vector comprising the dsRNA, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is for treating an inflammatory disease, such as arthritis, particularly rheumatoid arthritis (RA).
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, intra-articular or epidermal administration (e.g., by injection or infusion).
  • the active compound i.e., the dsRNA molecule
  • the dsRNA of the invention can be delivered by a cationic liposome delivery system.
  • compositions of the invention may also contain a pharmaceutically acceptable antioxidant.
  • pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium hydrogen sulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants such as ascorbic acid palmitate Ester, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc.; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA) ), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium hydrogen sulfate, sodium metabisulfite, sodium sulfite, etc.
  • oil-soluble antioxidants such as ascorbic acid palmitate Ester, butylated
  • compositions may also contain, for example, preservatives, wetting agents, emulsifying agents, and dispersing agents.
  • the prevention of the presence of microorganisms can be ensured by a sterilization procedure or by the inclusion of various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol sorbic acid, and the like.
  • various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol sorbic acid, and the like.
  • isotonic agents for example, sugars, polyols such as mannitol, sorbitol or sodium oxide.
  • Prolonged absorption of the injectable drug can be achieved by the addition of a delay absorbent such as monostearate and gelatin to the composition.
  • Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and powders for the preparation of sterile injectable solutions or dispersions.
  • the use of such media and agents for pharmaceutically active substances is well known in the art.
  • Conventional media or agents may be in the pharmaceutical compositions of the present invention, except in the range which is incompatible with the active compound. Additional active compounds can also be incorporated into the compositions.
  • compositions must generally be sterile and stable under the conditions of manufacture and storage.
  • the compositions can be formulated as solutions, microemulsions, liposomes or other ordered structures suitable for high drug concentrations.
  • the carrier can be a solvent or dispersing agent containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants.
  • Sterile injectable solutions can be prepared by incorporating the active compound in a suitable amount in a suitable solvent, and, if necessary, one or a combination of the ingredients listed above, followed by sterile microfiltration.
  • dispersing agents are prepared by incorporating the active compound into a sterile vehicle which may contain a base dispersion medium and the other ingredients listed above.
  • the preferred preparation methods are vacuum drying and lyophilization (lyophilization), a solution which is pre-sterilized in such a manner as a powder of the active ingredient plus any additional desired ingredients.
  • the amount of active ingredient that can be combined with the carrier materials in a single dosage form will vary depending upon the subject being treated and the particular mode of administration.
  • the amount of active ingredient that can be combined with the carrier materials in a single dosage form is generally the amount of the composition that produces the therapeutic effect. Typically, this amount will range from about 0.01% to about 99% active ingredient, for example from about 0.1% to about 70%, for example from about 1% to about 30%, by weight of the active ingredient, with pharmaceutically acceptable The carriers are combined.
  • the dosage regimen can be adjusted to provide the optimal desired response (eg, a therapeutic response). For example, a single bolus can be administered, several separate doses can be administered over time, or the dose can be reduced or increased proportionally as needed for an emergency condition of the treatment condition. It is especially advantageous to formulate the parenteral compositions in dosage unit form for ease of administration and uniformity.
  • Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for the subject to be treated; each unit contains a predetermined amount of active compound which is calculated to produce the active compound in association with the required pharmaceutical carrier. The desired therapeutic effect.
  • the specific description of the dosage unit form of the present invention is limited to and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) inherent in the art for formulating such sensitivity for treating an individual. Limitation of active compounds.
  • the dosage may range from about 0.0000001 to 100 mg/kg of recipient body weight.
  • An exemplary treatment regimen may be once weekly, once every two weeks, once every three weeks, once every four weeks, once a month, once every two months, once every three months, once every four months, every time Once every 5 months, once every 6 months, once every 7 months, once every 8 months, once every 9 months, every 10 months, every 11 months, even every 12 months, or from The initial dosing interval is slightly shorter (eg, once a week to once every three weeks) and the post-dosing interval is lengthened (eg, once a month to even once every 12 months).
  • the actual dosage level of the active ingredient in the pharmaceutical compositions of the present invention may be varied to achieve an amount of active ingredient which is effective to achieve the desired therapeutic response to a particular patient, composition and mode of administration without toxicity to the patient.
  • the selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular composition of the invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of treatment, and the particularity of the application.
  • Other drugs, compounds and/or materials to which the composition is used in combination the age, sex, weight, condition, general health and medical history of the patient being treated, and similar factors well known in the medical arts.
  • the present inventors designed more than 200 siRNA molecules directed against the CKIP-1 gene, and screened for siRNA molecules capable of significantly inhibiting CKIP-1 expression (Examples 1-3).
  • the CKIP-1 molecule of the present invention can significantly inhibit the expression of CKIP-1 protein in human osteoblasts (Example 5), and administration of the dsRNA can promote the expression of human osteoblast phenotype genes, thereby promoting Osteoblast differentiation (Examples 6 and 7).
  • the dsRNA of the invention is capable of inhibiting the expression of the pro-inflammatory cytokines IL-6, TNF-[alpha] and/or IL-17A (Examples 4 and 8).
  • the dsRNA of the present invention can significantly inhibit the expression of the pro-inflammatory cytokine IL-6.
  • TNF- ⁇ is mainly expressed by macrophages, synovial lining cells and activated T cells of inflamed joints.
  • TNF- ⁇ is one of the most pro-inflammatory cytokines and is capable of inducing the production of other pro-inflammatory factors such as IL-1 ⁇ , IL-6 and IL-8.
  • the IL-6 receptor neutralizing antibody in the CIA-induced process completely abolished the inflammatory response, indicating that IL-6 plays an important role in the process of causing arthritis.
  • the dsRNA targeting CKIP-1 of the present invention is capable of inhibiting the expression of pro-inflammatory cytokines and thus can be used to treat inflammation.
  • the ability of the dsRNAs of the present invention to target CKIP-1 to inhibit inflammation is particularly advantageous in the treatment of arthritis, particularly rheumatoid arthritis. Because in RA, the early symptoms are mainly joint inflammation, and bone destruction occurs after several years (called “late bone destruction”).
  • the CKIP-1-targeting dsRNA of the present invention is capable of inhibiting both inflammation and bone damage, and thus can be advantageously used in various stages of RA treatment, and is not limited to late bone destruction.
  • the invention provides a method of treating arthritis, particularly rheumatoid arthritis, in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a dsRNA molecule of the invention or the invention An expression vector or a pharmaceutical composition of the invention.
  • the present invention provides a dsRNA of the present invention or an expression vector of the present invention or a pharmaceutical composition of the present invention, in the preparation of a medicament for treating arthritis, particularly rheumatoid arthritis, in a subject in need thereof use.
  • Arthritis that can be treated by the dsRNA molecules of the invention or the expression vector of the invention or the pharmaceutical composition of the invention includes, but is not limited to, rheumatoid arthritis, osteoarthritis, idiopathic arthritis, ankylosing spondylitis, silver shavings Disease arthritis, infectious arthritis, juvenile arthritis, reactive arthritis, gouty arthritis, etc.
  • dsRNA of the invention or the expression vector of the invention or the pharmaceutical composition of the invention may also be used in combination with additional therapeutic agents for the treatment of arthritis, particularly rheumatoid arthritis.
  • additional therapeutic agents include, but are not limited to, non-steroidal anti-inflammatory drugs, hormones, anti-rheumatic drugs, and the like.
  • the invention provides a method of treating a proinflammatory cytokine (eg, IL-6, TNF-[alpha], and/or IL-17A) associated inflammatory disease in a subject in need thereof, comprising administering to the subject a treatment An effective amount of a dsRNA molecule of the invention or an expression vector of the invention or a pharmaceutical composition of the invention.
  • a proinflammatory cytokine eg, IL-6, TNF-[alpha], and/or IL-17A
  • the invention also provides a dsRNA of the invention or an expression vector of the invention or a pharmaceutical composition of the invention in the preparation of a pro-inflammatory cytokine (eg, IL-6, TNF-[alpha] and in a subject in need thereof / or IL-17A) use in drugs related to inflammatory diseases.
  • a pro-inflammatory cytokine eg, IL-6, TNF-[alpha] and in a subject in need thereof / or IL-17A
  • Inflammatory cytokines eg, IL-6, TNF-[alpha], and/or IL-17A
  • inflammatory diseases including, but not limited to, inflammatory bowel disease, inflammation caused by infection, inflammation caused by injury, respiratory inflammation, cancer related sexual inflammation, etc.
  • the inflammatory diseases associated with the pro-inflammatory cytokines e.g., IL-6, TNF-[alpha], and/or IL-17A
  • arthritis such as the arthritis listed above, particularly rheumatoid arthritis.
  • proinflammatory cytokines eg, IL-6, TNF-[alpha], and/or IL-17A
  • associated inflammatory diseases include, but are not limited to, Systemic lupus erythematosus, Crohn's disease, psoriasis, colitis, ileitis, glomerulonephritis, asthma, dermatitis (including contact dermatitis and atopic dermatitis), vasculitis, chronic bronchitis, chronic prostatitis , appendicitis, pancreatitis, pelvic inflammatory disease, polymyositis, chronic obstructive pulmonary disease.
  • the dsRNA of the invention or the expression vector of the invention or the pharmaceutical composition of the invention may also be additionally used for the treatment of inflammatory diseases, in particular pro-inflammatory cytokines (eg IL-6, TNF- ⁇ and/or IL-17A)
  • cytokines eg IL-6, TNF- ⁇ and/or IL-17A
  • a therapeutic agent for a related inflammatory disease is used in combination.
  • the additional therapeutic agent is, for example, an inhibitor that targets TNF-[alpha], including but not limited to infliximab, etanercept, adalimumab, golimumab, and certolizumab; IL -6 blockers, including but not limited to Tocilizumab; IL-17A blockers, including but not limited to secukinumab.
  • the invention provides a method of treating a bone metabolism-related disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a dsRNA molecule of the invention or an expression vector of the invention or a medicament of the invention combination.
  • the invention also provides the use of a dsRNA of the invention or an expression vector of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament for use in a bone metabolism related disorder in a subject in need thereof.
  • the bone metabolism-related diseases include, but are not limited to, osteomalacia, osteopenia, osteolytic bone disease, renal osteopathy, osteogenesis imperfecta, bone destruction caused by cancer bone metastasis, and the like.
  • dsRNA of the present invention or the expression vector of the present invention or the pharmaceutical composition of the present invention can also be used in combination with an additional therapeutic agent for treating a disease associated with bone metabolism.
  • the invention provides a method of reducing the level of a pro-inflammatory cytokine (eg, IL-6, TNF-[alpha], and/or IL-17A) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount A dsRNA molecule of the invention or an expression vector of the invention or a pharmaceutical composition of the invention.
  • a pro-inflammatory cytokine eg, IL-6, TNF-[alpha], and/or IL-17A
  • the object is a human.
  • the dsRNA of the invention or the expression vector of the invention or the pharmaceutical composition of the invention is administered intra-articularly. In some embodiments, the dsRNA of the invention or the expression vector of the invention or the pharmaceutical composition of the invention is administered systemically.
  • the candidate siRNA pool was designed according to the homologous region of human CKIP-1 mRNA and monkey CKIP-1 mRNA sequence, and the off-target effect of candidate siRNA pool was analyzed comprehensively. Finally, the candidate siRNA sequence with high off-target score was eliminated by combining the seed region matching score.
  • 208 siRNA candidate sequences against CKIP-1 were obtained and synthesized. Eight unrelated NC sequences were also designed and synthesized as negative controls in screening assays. The synthetic 208-entry gene siRNA sequence, as well as the eight NC sequences, are shown in Figure 1.
  • Example 2 Screening of siRNAs that inhibit the expression of CIKP-1 by real-time quantitative PCR
  • hFOB cells human osteoblasts, purchased from the Chinese Academy of Sciences cell bank
  • siRNA transfection was performed at a cell density of about 70%.
  • 0.5 ⁇ l of Lipofectamine 2000 was diluted in 25 ⁇ l of serum-free and antibiotic-containing opti-MEM and mixed well.
  • 15 pmol RNA was diluted in 25 ⁇ l of anti-serum and antibiotic-free opti-MEM and mixed gently.
  • RNA Triangen MicroRNA Extraction Kit
  • reverse transcription Tekara Reverse Transcription Kit
  • qPCR detection full-scale gold qPCR kit
  • CIKP1-F GGAACCAACCTCTTGTGCTG
  • CIKP1-R GTCAACTTCTTGGGTGCCTG
  • GADPH-F CATGAGAAGTATGACAACAGCCT
  • GADPH-R AGTCCTTCCACGATACCAAAGT
  • siRNA sequence Normalized target gene relative expression si-TD037 0.472339 si-TD040 0.457801 Si-TD042 0.422001 si-TD044 0.398672 si-TD050 0.307432 Si-TD057 0.417976 Si-TD058 0.412397 si-TD060 0.250549 Si-TD061 0.314191 si-TD062 0.198302 si-TD064 0.46957 si-TD065 0.4389 si-TD066 0.30317 si-TD067 0.411764 Si-TD068 0.252114 si-TD070 0.281898 si-TD072 0.401834 si-TD074 0.220171 si-TD076 0.334746 si-TD078 0.318811 Si-TD080 0.23612 si-TD082 0.297076 si-TD084 0.356374 si-TD087 0.32098 si-TD089 0.238577 Si-TD093 0.367916 si-TD094 0.410962 si-TD096 0.
  • si-TD221 0.462056 si-TD224 0.490381 si-TD358 0.387057 si-TD362 0.288363 si-TD364 0.275357 si-TD370 0.445778 Si-TD372 0.459658 si-TD376 0.387624 si-TD378 0.295441 si-TD380 0.400417 si-TD443 0.396858 si-TD451 0.311861 si-TD480 0.460598 si-TD483 0.377209 si-TD508 0.476182 si-TD509 0.468754 si-TD577 0.424962 si-TD585 0.448536 si-TD587 0.410307 si-TD588 0.441516 si-TD596 0.497351 si-TD598 0.422082 si-TD600 0.487359 si-TD604 0.401307 si-TD607 0.375209 si-TD609 0.476541 si-TD611 0.457187 si-TD717 0.467227 si-TD718 0.45
  • Example 2 the candidate siRNA sequences screened in Example 2 were further characterized by dual luciferase assay.
  • the CKIP-1 CDS 1-652 sequence fragment was amplified by PCR using upstream and downstream primers with SacI and XhoI restriction sites and protective bases, respectively.
  • the amplified product was digested with SacI and XhoI and inserted into the pGP-miRGLO overexpression vector (see Figure 2) also digested with SacI and XhoI to obtain the pmirGlo-CDS1 vector: overexpressing the first segment of the CDS-1 gene CDS region 1
  • the sequence of -652 The sequence of -652.
  • the CKIP-1 CDS 653-1230 sequence fragment was amplified by PCR using upstream and downstream primers with SacI and XhoI restriction sites and protective bases, respectively.
  • the amplified product was digested with SacI and XhoI and inserted into the pGP-miRGLO overexpression vector (see Figure 2) also digested with SacI and XhoI to obtain the pmirGlo-CDS2 vector: overexpressing the second segment of the CDS-1 gene CDS region 653 The sequence of -1230.
  • 293T cells routinely cultured in DMEM medium (Gibco) containing 10% FBS (Gibco) (containing 1.5 mM L-glutamine, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin), saturated at 37 ° C 5% CO 2 Culture in a humidity incubator.
  • the medium in the 24-well plate that was laid the day before was removed, and the medium was added at 400 ⁇ l/well; after 20 minutes of standing time, the transfection mixture was separately added to the above 24-well plate, 100 ⁇ l/well, each Set 3 repeats, and set the Blank hole and Mock hole, shake the well plate and incubate in the incubator for 6 hours.
  • the transfection solution was removed, rinsed with PBS, culture medium was added, and the wells of NC-FAM were transfected to observe the transfection efficiency. Cells were harvested for dual luciferase assay 24 h after transfection.
  • Experimental materials and reagents include: Dual-Luciferase Reporter Assay System (Promega, E1960); PBS; 96-well white plate (corning cat. #3912); multi-label microplate reader (PerkinElmer EnSpire).
  • the medium in the cell plate to be tested is removed, the cultured cells are washed with PBS, and PBS is discarded. According to 100 ⁇ l/well, 1 ⁇ PLB was added, and the plate was gently shaken at room temperature and lysed for 15 min. The cell lysate was transferred to a small centrifuge tube and centrifuged at 3000 rpm for 3 min to remove cell debris. 30 ⁇ l of the supernatant was added to a 96-well white plate, and the substrate was added for detection according to the recommended procedure.
  • siRNAs had a good inhibitory effect on CKIP-1 expression in the dual luciferase assay: si-TD137, si-TD141, si-TD176 and si-7.
  • the results of the measurements are shown in Tables 3-6 and 3-6, respectively.
  • siRNA targeting CKIP-1 inhibits proinflammatory cytokine expression
  • the RAW264.7 mouse peritoneal macrophage cell line (purchased from the Chinese Academy of Sciences Cell Bank, Shanghai) was cultured in complete DMEM medium containing 10% fetal bovine serum, 100 U penicillin and streptomycin, and placed at 37 ° C with constant temperature carbon dioxide. (5%) incubator, culture overnight, until the cell fusion degree reached 70-80% for the experiment.
  • RNAi group small interfering RNA against CKIP-1 or its sense strand methoxy modification sequence prepared above was transfected into mouse macrophage as a drug treatment group (RNAi group), and the transfection reagent X-TremeGENE was used alone.
  • the siRNA transfection reagent purchased from Roche, Cat. No. 4476093001 was used to treat the above cells as a transfection reagent group (MOCK group), and each group was 3 in parallel, and at least 3 experiments were repeated.
  • MOCK group transfection reagent group
  • the final concentration of small interfering RNA was 30 nM.
  • LPS purchased from Sigma, product number L2630-10MG
  • LPS was added for 6 hours, and the supernatants of each group were collected to detect the secretion of pro-inflammatory cytokines, and the cells of each group were collected to detect the expression levels of pro-inflammatory cytokines mRNA.
  • the ELISA method was used to detect the inhibition efficiency of the secretion levels of TNF- ⁇ and IL-6 in the above cell supernatant, specifically: using Mouse TNF alpha ELISA (eBioscience, Cat. No. 88-7324-88) and Mouse IL-6 ELISA (eBioscience, Cat. No. 88-7064-88) kit, tested according to the instructions, and the concentration of TNF- ⁇ and IL-6 was calculated by plotting a standard curve.
  • Mouse TNF alpha ELISA eBioscience, Cat. No. 88-7324-88
  • Mouse IL-6 ELISA eBioscience, Cat. No. 88-7064-88
  • Cytokine inhibition efficiency is calculated as follows:
  • Cytokine inhibition efficiency [(LPS group - treatment group) / (LPS group - blank control group)] ⁇ 100%.
  • each candidate siRNA and its methylated modified form can inhibit the secretion of IL-6 and TNF- ⁇ by LPS-induced RAW264.7 mouse macrophages and inhibit the secretion of IL-6. Reach a significant level.
  • Real-time PCR was used to detect the levels of TNF- ⁇ and IL-6 mRNA in the harvested RAW264.7 cells. Specifically, the total RNA was extracted using TRIzol reagent (Invitrogen, Cat. No. 15596018), using TransScript All. -in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) (Full-Gold, Item No. AT341-02) kit for reverse transcription synthesis of cDNA, using fluorescent quantitative PCR to detect small siRNA induced by LPS Inhibition efficiency of IL-6 and TNF- ⁇ expression in murine peritoneal macrophages.
  • the GAPDH gene was used as an internal reference gene in the Real-time PCR method, and the primer sequences used are shown in Table 9.
  • nucleic acid inhibition efficiency is calculated according to the following equation:
  • siRNA inhibition efficiency [(LPS group cytokine gene copy number / LPS group GAPDH gene copy number - treatment group cytokine gene copy number / treatment group GAPDH gene copy number) / (LPS group cytokine gene copy number / LPS group GAPDH gene Copy number - blank control group cytokine gene copy number / blank control group GAPDH gene copy number)] ⁇ 100%
  • each candidate siRNA significantly inhibited LPS-induced IL-6 mRNA expression in mouse macrophages; there was no significant inhibition on TNF- ⁇ mRNA expression.
  • the siRNA targeting CKIP-1 of the present invention can inhibit the levels of the pro-inflammatory cytokines IL-6 and TNF- ⁇ , particularly IL-6, thereby inhibiting inflammation, particularly IL-6 and/or TNF- ⁇ . Inflammation, such as inflammation in RA.
  • Example 5 Inhibitory effect of siRNA targeting CKIP-1 on CKIP-1 protein expression
  • the human osteoblast cell line hFOB1.19 was purchased from the Chinese Academy of Sciences cell bank, and the culture solution was DMEM-F12 medium (purchased from Gibco) containing 10% fetal calf serum.
  • the human osteoblast cell line hFOB1.19 was transferred to a 24-well plate and cultured overnight to allow adherence.
  • the human osteoblast-like cell line hFOB1.19 was transfected with siRNA targeting CKIP-1 as a treatment group, and the above cells were transfected with a non-specific nucleic acid as a negative control group (NC group). Each group was 2 parallels and at least 3 experiments were repeated. When transfected into human osteoblast-like cells, the final concentration of nucleic acid was 20 ⁇ M. After 72 hours of transfection, cells were harvested for detection of CKIP-1 protein expression.
  • CKIP-1 protein in osteoblast-like cells was detected by immunoblotting.
  • the CKIP-1 antibody used for immunoblot detection was purchased from Santa Cruz Biotechnology (Cat. No. sc-376355), and the internal reference antibody was GADPH (purchased from Santa Cruz Biotechnology, Inc., sc-166574).
  • nucleic acid inhibitory activity [1 - (light intensity value of the immunoblot band of the treatment group CKIP-1 protein / light intensity value of the immunoblotting band of the treatment group GAPDH protein) / (light intensity value of the CKIP-1 protein immunoblot band in the control group / light intensity value of the immunoblotting band of the control GAPDH protein)] ⁇ 100%.
  • ALP, COL1A1 and OPN were expressed in the early stage of osteoblast differentiation, and BSP and OC began to express in the mature function stage of osteoblasts. At 72h, the expression levels of ALP, COL1, OPN, BSP and OC in si-7 group were significantly higher than those in NC group.
  • the experimental results indicate that the siRNA targeting CKIP-1 of the present invention can promote the expression of the hFOB1.19 phenotype gene of human osteoblast cell line, thereby promoting osteoblast differentiation.
  • Example 7 Effect of siRNA targeting CKIP-1 on mineralization deposition rate of bone matrix
  • Calcium deposition is a key functional mineralization marker for mature osteoblasts during osteoblast formation in vitro.
  • human osteoblast-like cell line hFOB1.19 was transfected with siRNA targeting CKIP-1 as a treatment group, and the above cells were transfected with a non-specific nucleic acid as a negative control group (NC group). At the time of transfection, the final concentration of the nucleic acid was 20 ⁇ M. The frequency of interstitial transfection was once a week, with 4 parallels in each group.
  • Calcium deposition in human osteoblast-like cell line hFOB 1.19 was determined by calcium staining after 7, 14 and 21 days of the first transfection.
  • the measurement results are shown in Table 14. After 21 days of human osteoblast-like cells transfected for the first time, the calcium deposition in the treated group was significantly higher than that in the NC group, which at the functional level demonstrated that the siRNA of the present invention can promote differentiation of human pre-osteoblasts into mature osteoblasts.
  • Example 8 Evaluation of siRNA in vivo activity using a mouse CIA model
  • CIA collagen-induced arthritis
  • the specific method is as follows: an appropriate amount of 2 mg/mL bovine type II collagen is mixed with an equal amount of incomplete Freund's adjuvant, and fully emulsified, and the emulsified mixture is subcutaneously injected with 100 ⁇ g of type II collagen/mouse in the base of the tail. After 21 days, 50 ⁇ g of type II collagen was added/subcutaneously only once under the root of the tail.
  • the 5-level semi-quantitative scoring standard was used as the criterion for the clinical severity of arthritis: 0: no redness; 1: erythema with mild swelling, and limited to the middle of the foot or the ankle joint; 2: mild swelling from the ankle to the middle of the foot Extension; 3: moderate swelling from the ankle joint to the tibial joint; 4: ankle joint, foot and toe severe swelling.
  • Animals were randomly assigned to visually assess the severity of both hind limbs with a mean score of 1 point for each group of animals: vehicle group, intra-articular cavity injection of blank liposomes; NC (negative control) group, ankle joint injection Liposomes with negative control sequences; treated group, intra-articular cavity injection of liposomes containing Si-7, Si-137, Si-141 or Si-176; positive control group, positive drug Eceercept (Etanercept) Each containing 12.5mg of active ingredient was purchased from Shanghai CITIC Guojian Pharmaceutical Co., Ltd.).
  • the animals in each group were given bilateral ankle joint injections on the 0th, 7th, 14th, 21st, 28th and 35th day of the group.
  • the dose was 4 ⁇ g siRNA/5 ⁇ l liposome/ ankle joint.
  • the positive drug Yisaipu was administered by subcutaneous injection at a dose of 7.5 mg/kg body weight.
  • mice The scores of the ankle joint swelling of the mice were observed and recorded from the day of enrollment, and were recorded twice a week. The scores of the two hind limbs were statistically analyzed. The clinical scoring results are shown in Figure 7 and Table 15 below. At the same time, the mouse body weight was recorded once a week, and the results are shown in Fig. 8 and Table 16.
  • CIA model mice were sacrificed, the leg skins were cut with scissors, and the ankle joints were exposed. The lower part of the knee joint was cut with a rongeur, cooled by liquid nitrogen and transferred to an enzyme-free tube using TRIzol reagent (Invitrogen, Cat. No. 15596018). Extract total cellular RNA.
  • the cDNA was reverse transcribed using the TransScript All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) kit (full-size gold, Cat. No. AT341-02) kit.
  • the inhibitory efficiency of si-7, si-137, si-141 and si-176 on the expression of CKIP-1, IL-6, TNF- ⁇ and IL-17A mRNA in CIA mouse model was detected by real-time PCR.
  • the primers used for IL-6, TNF- ⁇ , and the internal reference gene GAPDH are the same as above.
  • the CKIP-1 and IL-17A primer sequences are shown in Table 17:
  • Inhibition efficiency [(carrier group cytokine gene copy number / vector group GAPDH gene copy number - treatment group cytokine gene copy number / treatment group GAPDH gene copy number) / (carrier group cytokine gene copy number / vector group GAPDH gene copy Number-normal control cytokine gene copy number/normal control group GAPDH gene copy number)] ⁇ 100%
  • si-7-OMe, si-137-OMe, si-141-OMe, and si-176-OMe significantly inhibited CKIP-1, IL-6, TNF- ⁇ , IL in CIA mouse joint tissues.
  • -17A mRNA expression, inhibition rate was greater than 50%, and the inhibition of CKIP-1 mRNA was stronger than the positive drug Yisaipu (41.37%).
  • si-137 showed a stronger inhibitory effect on the pro-inflammatory factors IL-6, TNF- ⁇ , IL-17A mRNA, and the inhibition was stronger than the positive drug Yisaipu. It is shown that the siRNA of the present invention is effective for inhibiting inflammation in RA.
  • MicroCT was performed using ScancovivaCT 40.
  • the mouse hind paws were placed in a Micro CT sample tube for 3D CT scan reconstruction. After the scan is completed, the three-dimensional microstructure of the trabecular bone is analyzed and the spatial structure parameters of the trabecular bone are collected.
  • mice The hind limbs of the mice were fixed in 4% formaldehyde solution, and the EDTA was decalcified and embedded in paraffin. Serial sections were taken for HE staining to examine joint pathological changes and bone erosion.
  • each siRNA-administered group played an active role in improving inflammation and bone damage in the rheumatoid arthritis model and delaying the progression of the disease, reflecting a good therapeutic effect.
  • the experiment used 3-6 years old female normal cynomolgus monkey.
  • the collagen-induced arthritis modeling method was referred to the relevant literature.
  • the cynomolgus monkeys were immunized with bovine type II collagen on day 0 and day 21, respectively.
  • the drug is administered after the onset of the disease, and the administration form is local administration of the joint.
  • Small nucleic acids employ liposome delivery systems.
  • the groups were as follows: carrier group, intraocular cavity injection of blank liposome; NC (negative control) group, intra-articular cavity injection of liposome carrying negative control sequence; treatment group, joint cavity injection containing Si-7, Si- 137, Si-141 or Si-176 liposome; positive control group, the application of positive drug Yisaipu (Etanercept, purchased from Shanghai CITIC Guojian Pharmaceutical Co., Ltd.). Three animals in each group were injected intra-articularly once a week for 6 weeks.
  • MicroCT, pathology, bone morphometry, and the like are similar to mouse experiments.
  • the siRNA-administered group showed a good therapeutic effect on improving the condition, especially reducing bone damage and maintaining bone function, in the rheumatoid joint model.

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Abstract

L'invention concerne une molécule d'ARN double brin ciblant CKIP-1 et son utilisation dans le traitement d'une maladie inflammatoire comprenant la polyarthrite rhumatoïde.
PCT/CN2018/104552 2017-09-07 2018-09-07 Molécule d'arn double brin ciblant ckip-1 et son utilisation Ceased WO2019047914A1 (fr)

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US16/644,977 US11155819B2 (en) 2017-09-07 2018-09-07 Double-stranded RNA molecule targeting CKIP-1 and use thereof
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